Wild-type α2,6-sialyl transferases have been isolated from marine bacteria, such as Photobacterium damselae JT0160 (U.S. Pat. Nos. 5,827,714, 6,255,094, Yamamoto et al. J. Biochem. 123, 94 (1998)), and subsequently from Photobacterium sp. JT-ISH-224 (U.S. Pat. Nos. 7,993,875, 8,187,838, Tsukamoto et al. J. Biochem. 143, 187 (2008)) and subsequently from P. leiognathi JT-SHIZ-145 (U.S. Pat. Nos. 8,187,853, 8,372,617, Yamamoto et al. Glycobiology 17, 1167 (2007)) and finally from P. leiognathi JT-SHIZ-119 (US 2012/184016, Mine et al. Glycobiology 20, 158 (2010)). The α2,6-sialyl transferase from P. leiognathi JT-SHIZ-119 and the truncated α2,6-sialyl transferase from Photobacterium damselae JT0160 have been found to also have sialidase activity (US 2012/0184016, Mine et al. Glycobiology 20, 158 (2010), Cheng et al. Glycobiology 20, 260 (2010)). However, these wild-type α2,6-sialyl transferases have not been used or entirely suitable for making sialylated oligosaccharides, particularly sialylated human milk oligosaccharides (HMOs).
Mutants of such enzymes have therefore been sought, preferably having increased regioselectivity and/or increased thermostability, in particular for the enzymatic synthesis of sialylated oligosaccharides, especially sialylated HMOs.